Release of glucagon, a principal hormone underlying the normal counter-regulatory response (CRR) to acute hypoglycemia is largely absent in type 1 diabetes mellitus (T1DM). In its absence, other circulating CRR hormones, such as epinephrine, take on primary importance in restoring plasma glucose to basal levels. However, epinephrine release in response to hypoglycemia is also attenuated in T1DM, making diabetic patients particularly prone to asymptomatic hypoglycemia that, if untreated, can lead to coma and death. This defect has been minimally studied at the level of the adrenal medulla (AM) which produces most of all circulating epinephrine in the body. Chromaffin cells in the AM synthesize epinephrine and release it when activated by the central nervous system or other circulating hormones. Our preliminary data show that muscarinic acetylcholine receptor (mAchR) signaling is impaired in adrenomedullary chromaffin cells isolated from streptozocin (STZ)-treated diabetic rats and that type 3 mAchR, a predominant receptor subtype in the adrenal medulla, is internalized to a much greater degree in diabetic chromaffin cells than in controls. These data led me to hypothesize that internalization of adrenomedullary mAchR in T1DM reduces epinephrine release in response to hypoglycemia. To test this hypothesis, I plan to: (1) Determine the time after the on-set of diabetes when this defect occurs, the extent of the reduction, the specificity of the deficit and the involvement of diabetic hyperglycemia and hypoinsulinemia in its development. The STZ model of diabetes will be used in these studies. In vivo experiments will be performed to study the development of reduced adrenomedullary response to hypoglycemic and non-hypoglycemic stimuli during T1DM. Isolated adrenal glands and enzymatically- isolated chromaffin cells will be used to evaluate the contributions of nicotinic and muscarinic acetylcholine signaling and adrenal epinephrine content to this deficit. (2) Investigate the functional properties of acetylcholine receptors in adrenomedullary chromaffin cells during STZ-induced diabetes by measuring mRNA, protein levels, cellular localization and functional properties of nicotinic and muscarinic acetylcholine receptor subtypes in chromaffin cells from diabetic animals. The results of the proposed experiments will establish the cellular basis for the adrenal medullary defect in epinephrine release that contributes to the reduced CRR in T1DM. Health relevance: This project seeks to investigate the cellular basis for defective regulation of the body's response to low blood glucose in diabetic patients. This defect limits insulin-based therapies. PUBLIC HEALTH RELEVANCE: This project seeks to investigate the cellular basis for defective regulation of the body's response to low blood glucose in diabetic patients. This defect limits insulin-based therapies.